Dihydrolipoamide dehydrogenase (E3) is a shared component of the three alpha-keto acid dehydrogenase complexes (involved in the oxidation of pyruvate, alpha-ketoglutarate and branched-chain alpha-keto acids) and glycine synthase (involved in glycine oxidation). E3-deficient patients develop severe neurologic disabilities. E3 catalyzes the reoxidation of the dihydrolipoyl moiety of the dihydrolipoyl acyltransferase (E2) component of the complexes and the H protein of glycine synthase. E3 binds non- covalently to the peripheral subunit-binding domain in the E2 component from most species. In eukaryotic pyruvate dehydrogenase complex (PDC), E3 binds to an E3-binding protein (E3BP). The long- term goal of this proposal is to continue a deeper understanding of the subunit-subunit interactions of E3 with E3BP in mammalian PDC, transcriptional regulation of the E3 and E3BP genes and characterization of E3 deficiency using a mouse model. Based on our recent findings, the five specific aims of this proposal are: (i) to develop an E3-deficient mouse model, (ii) to investigate the impairment on brain development and oxidative metabolism of various fuels, (iii) to investigate the subunit-subunit interactions between human E3 with E3BP of PDC, (iv) to determine the genomic structure of the human E3BP gene and its transcriptional regulation, and (v) to characterize a novel transcription factor of the murine E3 gene. Using the combined approaches of homologous recombination and chimeric gene transfer technologies we propose to develop a mouse strain with less than 20% of control E3 activity. Structural impairment in the developing brain will be determined using histological and immunocytochemical analyses. Using recombinant human E3s and E3BPs (wild-type and site-specific mutants), the sites of interaction between these two proteins will be characterized. Using molecular biology techniques, the genomic organization and the proximal promoter region of the human E3BP gene will be characterized. To identify a novel transcription factor (upstream direct repeat element binding protein) for the murine Dld gene, we propose to purify this protein. From its partial amino acid sequence a cloning strategy will be developed to isolate a mouse cDNA clone for analysis. This multi-faceted approach is designed to enhance our understanding of the structure- function relationships of E3 and E3BP interactions, transcriptional regulation of the E3 and E3BP genes, and time-dependent pathological changes in developing brain of E3-deficient mice.